A complete experimental description of the mechanism of protein folding requires determination of the distribution of microscopic folding pathways. This information is only available from experiments on single molecules. As a first step toward monitoring the folding of individual molecules we have investigated the folding equilibrium of a small cold shock protein from the hyperthermophilic bacterium Thermotoga maritima (CspTm) using single molecule Forster resonance energy transfer (FRET). CspTm exhibits two-state thermodynamics and kinetics and is tolerant to structural modifications, making it a suitable protein for developing single molecule methods. Donor and acceptor dyes were attached to cysteine residues introduced at the amino and carboxy termini of the recombinantly expressed protein. In equilibrium experiments the FRET efficiency was determined for single molecules freely diffusing through the focal volume illuminated with an argon ion laser. The folding equilibrium constant and the mean value and width of the FRET efficiency distributions were determined as a function of denaturant concentration. Comparison of the equilibrium data with the results of ensemble experiments provides new insights into the distribution of structures in both the native and denatured ensembles. These include counting the number of sub-populations in the solution and therefore the number of thermodynamic states, measurement of the size of the protein in the denatured state from the end to end distance, and limits on the polypeptide reconfiguration time in the denatured state. Using Kramers theory for barrier crossing processes in the high friction limit, the last quantity yields the minimum height of the free energy barrier that separates the folded and unfolded states, and provides a new kind of critical theoretical test of both simulations and theories of protein folding.